Hypoxia-induced phase separation of ZHX2 drives 3D chromatin remodeling and cancer metastasis (ATAC-Seq). Hypoxia-induced phase separation of ZHX2 drives 3D chromatin remodeling and cancer metastasis (ATAC-Seq)

Hypoxia and dysregulation of three-dimensional (3D) chromatin architecture can both activate oncogenic transcriptomic profiles, thus contributing to tumor malignancy. But how hypoxia regulates 3D chromatin architecture remains unknown. Here, we find that the transcription factor, zinc fingers and homeoboxes 2 (ZHX2), generates liquid-liquid phase separation (LLPS) under hypoxia, thus promoting its occupancy on chromatin and activating transcription for a cluster of oncogenes, that is enriched by metastatic genes distinct from targets of hypoxia-inducible factor (HIF) and pathologically relevant to breast cancer. Mechanistically, hypoxia induces the LLPS of ZHX2 via a proline-rich intrinsically disordered region (IDR) in the nuclear localization sequence (NLS), thereby enhancing the phosphorylation of ZHX2 at S625 and S628 that incorporates CCCTCbinding factor (CTCF) in condensates to reorganize chromatin looping, consequently resulting in oncogenic super-enhancer formation and breast cancer metastasis. This fundamental mechanism provides significant insight into oncogene activation and suggests a phase separation-based therapeutic strategy for cancer. Overall design: We conducted ATAC-seq experiments in T47D cells under normoxia, hypoxia, and hypoxia with ZHX2 knockdown (KD).

缺氧与三维（3D）染色质结构失调，均可激活致癌性转录组特征，进而推动肿瘤恶性进展。但缺氧如何调控三维染色质结构，目前仍未明确。本研究发现，转录因子锌指与同源框蛋白2（zinc fingers and homeoboxes 2, ZHX2）可在缺氧条件下发生液-液相分离（liquid-liquid phase separation, LLPS），借此提升其在染色质上的结合占有率，并激活一组致癌基因的转录；该致癌基因簇富含转移相关基因，其调控靶点不同于缺氧诱导因子（hypoxia-inducible factor, HIF）的靶标，且与乳腺癌的病理进程密切相关。从机制层面来看，缺氧通过锌指与同源框蛋白2核定位序列（nuclear localization sequence, NLS）中富含脯氨酸的内在无序区域（intrinsically disordered region, IDR）诱导其发生液-液相分离，进而增强锌指与同源框蛋白2在S625与S628位点的磷酸化；该磷酸化事件可募集CCCTC结合因子（CCCTC-binding factor, CTCF）进入其凝聚体，从而重排染色质环，最终促成致癌超级增强子的形成与乳腺癌转移。这一基础性机制为致癌基因激活机制提供了重要见解，同时为基于相分离的癌症治疗策略提供了新方向。整体实验设计：本研究分别在常氧、缺氧以及缺氧联合锌指与同源框蛋白2敲低（knockdown, KD）的T47D细胞中开展了ATAC-seq实验。